LFA-1 and Mac-1 define characteristically different intralumenal crawling and emigration patterns for monocytes and neutrophils in situ

Abstract

To exit blood vessels, most (∼80%) of the lumenally adhered monocytes and neutrophils crawl toward locations that support transmigration. Using intravital confocal microscopy of anesthetized mouse cremaster muscle, we separately examined the crawling and emigration patterns of monocytes and neutrophils in blood-perfused unstimulated or TNF-α-activated venules. Most of the interacting cells in microvessels are neutrophils; however, in unstimulated venules, a greater percentage of the total monocyte population is adherent compared with neutrophils (58.2 ± 6.1% versus 13.6 ± 0.9%, adhered/total interacting), and they crawl for significantly longer distances (147.3 ± 13.4 versus 61.8 ± 5.4 μm). Intriguingly, after TNF-α activation, monocytes crawled for significantly shorter distances (67.4 ± 9.6 μm), resembling neutrophil crawling. Using function-blocking Abs, we show that these different crawling patterns were due to CD11a/CD18 (LFA-1)- versus CD11b/CD18 (Mac-1)-mediated crawling. Blockade of either Mac-1 or LFA-1 revealed that both LFA-1 and Mac-1 contribute to monocyte crawling; however, the LFA-1-dependent crawling in unstimulated venules becomes Mac-1 dependent upon inflammation, likely due to increased expression of Mac-1. Mac-1 alone was responsible for neutrophil crawling in both unstimulated and TNF-α-activated venules. Consistent with the role of Mac-1 in crawling, Mac-1 block (compared with LFA-1) was also significantly more efficient in blocking TNF-α-induced extravasation of both monocytes and neutrophils in cremaster tissue and the peritoneal cavity. Thus, mechanisms underlying leukocyte crawling are important in regulating the inflammatory responses by regulating the numbers of leukocytes that transmigrate.

Figure 1. The majority of monocytes but not neutrophils adher and crawl in uninflamed venules

The interactions of monocytes (stained for F4/80, 1.5µg/mouse, i.v) and neutrophils (stained for GR-1, 1.5µg/mouse, i.v) with the vessel wall were quantified in unstimulated venules. (A) Interacting cells were defined as all cells in immediate contact with the vessel wall. Field of view is ~300µm length. (B) The fraction of leukocytes that exhibited crawling out of total number of adhered cells. For A and B n=11 venules, in 4 mice (C) Representative images of the fields of view of selected venules where either monocytes (upper panel) or neutrophils (bottom panel) were immunofluorescently labeled. White arrows indicate the adhered leukocytes (confirmed from analysis of the movie from which the frame was extracted). The bar is 15µm. While the majority of neutrophils exhibit rolling behavior in unstimulated venules, the majority of monocytes were found adhered or crawling under these conditions. (D) The round shape of a representative rolling neutrophil, and a flattening neutrophil which is transitioning from adhesion to crawling, as captured by electron microscopy (upper panels, the bar is 1.8µm), and in-situ bright field microscopy (bottom panel, the bar is 10µm).

Figure 2. Monocyte crawling patterns become neutrophil-like upon TNFα activation

In separate experiments monocytes and neutrophils were stained for F4/80, and GR-1 respectively, (1.5µg/mouse, i.v). (A) The number of adhered cells/field of view. * Significantly different from appropriate control group (p<0.05), ** (p<0.01). Time lapsed microscopy (×90) was used to track crawling leukocytes. (B) The fraction of crawling leukocytes out of total adhered. For A and B n=11 venules, 4 mice. (C–E) the crawling velocities (C), the crawling distance (D) and the confinement ratio (as defined in Methods) were quantified. n=84–125 leukocytes, 4 mice. ** Significantly different from all other groups (p<0.01) (F) Representative monocyte crawling trajectories (n=10) from unstimulated (upper panel) and TNFα activated (lower panel) venules. All starting positions were aligned to the same origin. Black arrow indicates flow direction. Axes, distance in micrometers. (G) Images of time-lapse series over 30 minutes of real time acquisition were summed to display the paths (red dotted lines) of 3 crawling monocytes (upper panel) versus 3 crawling neutrophils (lower panel) in representative unstimulated venules over 40 minutes time period. While neutrophils maintain similar crawling patterns in both unstimulated and TNFα activated venules, monocytes switch from long distance, unconfined crawling to neutrophil-like, short distance and more direct crawling.

Figure 3. Roles for LFA-1 and Mac-1 in mediating monocyte and neutrophil crawling in unstimulated and inflamed venules

In separate experiments monocytes and neutrophils were stained for F4/80, and GR-1 respectively, (1.5µg/mouse, i.v) and were treated with either non-specific IgGs, Mac-1 blocking Ab, LFA-1 blocking Ab, or a combination of both (50µg/mouse, i.v). The effects of these blocking Abs on the ability of both monocytes and neutrophils to adhere and crawl were quantified in unstimulated tissue (A) and (C), and tissue that was treated with TNFα four hours prior to Ab administration (B) and (D). The effects of both IgG2a (a control isotope for LFA-1) and IgG2b (control isotope for Mac-1) on leukocyte adhesion and crawling were tested. As no significant differences were found between the two non-specific IgGs for each condition, for simplicity all data are presented against IgG2b. While neutrophil crawling was Mac-1 dependent in both unstimulated and TNFα activated venules, monocytes primarily used LFA-1 in unstimulated and Mac-1 in TNFα activated venules. For all groups n=11 venules, 4–6 mice. * Significantly different from IgG treatment within the same treatment group (p<0.05), **/^^ (p<0.01). ^&Significantly different from each other (p<0.01).

Figure 4. LFA-1 mediated crawling is for characteristically longer distances than that mediated by Mac-1

In separate experiments monocytes and neutrophils were stained for F4/80, and GR-1 respectively, (1.5µg/mouse, i.v) and were treated with either non-specific IgG, Mac-1 blocking Ab, LFA-1 blocking Ab, or a combination of both (50µg/mouse, i.v). The effects of these blocking Abs on the crawling distance and the crawling velocity of both monocytes and neutrophils were quantified in unstimulated tissue (A) and (C), and tissue that was treated with TNFα four hours prior to Ab administration (B) and (D). The effects of both IgG2a (a control isotope for LFA-1) and IgG2b (control isotope for Mac-1) on leukocyte adhesion and crawling were tested. As no significant differences were found between the two non-specific IgGs for each condition, for simplicity all data are presented against IgG2b. LFA-1 was able to sustain significantly longer crawling distances than Mac-1. For all groups n=11 venules, 4–6 mice. */^& Significantly different from each other (p<0.05), **/^^ (p<0.01).

Figure 5. In situ administration of TNFα increases the expression of Mac-1 but not LFA-1 on circulating monocytes and neutrophils

Flow cytometry was used to measure the surface expression of Mac-1 and LFA-1 on monocytes and neutrophils isolated from mouse circulation under baseline and TNFα-activated conditions. Isolated leukocytes were double labeled with either anti-F4/80 or Gr-1 (10µg/ml) in combination with either CD11a or CD11b (20µg/ml) and the number of antibody binding sites per cell was established as defined in Methods. Both monocytes and neutrophils isolated from TNFα treated mice significantly increased Mac-1 expression, however the expression of LFA-1 on monocytes was significantly lower compared to unstimulated tissue, but remained unchanged on neutrophils. n=3 mice. ** Significantly different from each other (p<0.01).

Figure 6. Mac-1 plays a more prominent role than LFA-1 in leukocyte TEM in cremaster venules

Leukocyte TEM in cremaster tissue in response to TNFα treatment was quantified in the abscence (−), or in the presence of either, a non-specific IgG (data not shown), LFA-1 or Mac-1 blocking Abs. Monocyte and neutrophils markers F4/80 and GR-1 respectively (1.5µg/mouse), as well as all Ab solutions (50µg/mouse) were administered via tail vein injection 10 minutes prior to the injection of TNFα (100ng in 50µl saline, ip.) and 4 hours prior to observations. (A) The total number of all leukocytes transmigrated into the extravascular tissue (100000 µm²) was quantified using bright field microscopy. (B) In separate experiments transmigrated monocytes and neutrophils stained for F4/80 and GR-1 respectively were counted in the extravascular tissue (100000 µm²) under the specified conditions, using fluorescence illumination. For all groups n=10–13 venules, 4–6 mice. ** Significantly different from TNFα alone (p<0.01). (C) Representative images (Z-stack projection of microvessels confocally scanned from the upper wall through the middle of the vessel) of neutrophils (upper panels) and monocytes (bottom panels) within the blood vessels (outlined with white lines) and in the surrounding tissue under the conditions specified on each panel. The bar is 15µm. Blockade of both LFA-1 and Mac-1 significantly reduced leukocyte TEM, but Mac-1 block was more effective (as quantified in panel B).

Figure 7. Total leukocyte and specifically neutrophil emigration into the peritoneal cavity is primarily Mac-1 dependent

Selected mice were injected with either saline (200µl), or Mac-1 blocker, LFA-1 blocker or non-specific IgG Abs (50µg/mouse in 200µl saline) 10 minutes prior to the injection of TNFα (100ng in 50µl saline, ip.). Four hours later peritoneal cavities of anesthetized mice were lavaged and white cells were recovered. (A) The number of total leukocytes was counted using a hemocytometer. (B) Neutrophil counts were determined on 100 µL cytospins stained with Diff-Quik and presented as percent total population. For all groups in A and B n=3–4 mice. **/^& Significantly different from each other (p<0.01). (C–F) Representative images of cytospins stained with Diff-Quik illustrate the dominance of mononuclear leukocytes in unstimulated peritoneal cavity (C), influx of neutrophils following TNFα treatment (D), which was prevented by Mac-1 blocking Ab (E), but not LFA-1 (F). White arrows point to the mononuclear leukocytes and black arrows point to neutrophils. The effects of both IgG2a (control isotope for LFA-1) and IgG2b (control isotope for Mac-1) on leukocyte adhesion and crawling were tested. As no significant differences were found between the two non-specific IgGs for each condition, for simplicity all data are presented against IgG2b. The bar is 12µm. Consistent with the cremaster observations, Mac-1 plays a more prominent role in leukocyte TEM compared to LFA-1.